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Open Access Available online http://arthritis-research.com/content/9/2/R24 Page 1 of 3 (page number not for citation purposes) Vol 9 No 2 Research article CTLA-4 +49A/G and CT60 gene polymorphisms in primary Sjögren syndrome Jacques-Eric Gottenberg 1 , Pascale Loiseau 2 , Mariam Azarian 2 , Chun Chen 2 , Nicolas Cagnard 3 , Eric Hachulla 4 , Xavier Puechal 5 , Jean Sibilia 6 , Dominique Charron 2 , Xavier Mariette 1 and Corinne Miceli-Richard 1 1 Rhumatologie, Institut Pour la Santé et la Recherche Médicale (INSERM) U802, Université Paris-Sud 11, Hôpital Bicêtre, 78 rue du Général Leclerc, Assistance Publique-Hôpitaux de Paris (AP-HP), 94275 Le Kremlin Bicêtre, France 2 INSERM 396, Immunologie et Histocompatibilité, Hôpital Saint-Louis, 1 avenue Claude-Vellefaux, AP-HP, 75475 Paris cedex 10, France 3 Institut Cochin, Unité de biologie moléculaire, Hôpital Cochin, 27 rue du Faubourg Saint-Jacques, 75679 Paris cedex 14, France 4 Médecine Interne, Hôpital Claude Huriez, 2 avenue Oscar Lambret, 59000 Lille, France 5 Rhumatologie, Hôpital du Mans, 194 avenue Rubillard, 72037 Le Mans, France 6 Rhumatologie, Hôpital Hautepierre, 1 avenue Molière, 67098 Strasbourg, France Corresponding author: Xavier Mariette, xavier.mariette@bct.aphp.fr Received: 23 Nov 2006 Revisions requested: 9 Jan 2007 Revisions received: 5 Feb 2007 Accepted: 6 Mar 2007 Published: 6 Mar 2007 Arthritis Research & Therapy 2007, 9:R24 (doi:10.1186/ar2136) This article is online at: http://arthritis-research.com/content/9/2/R24 © 2007 Gottenberg et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract CTLA-4 encodes cytotoxic T lymphocyte-associated antigen-4, a cell-surface molecule providing a negative signal for T-cell activation. CTLA-4 gene polymorphisms have been widely studied in connection with genetic susceptibility to various autoimmune diseases, but studies have led to contradictory results in different populations. This case-control study sought to investigate whether CTLA-4 CT60 and/or +49A/G polymorphisms were involved in the genetic predisposition to primary Sjögren syndrome (pSS). We analysed CTLA-4 CT60 and +49A/G polymorphisms in a first cohort of 142 patients with pSS (cohort 1) and 241 controls, all of Caucasian origin. A replication study was performed on a second cohort of 139 patients with pSS (cohort 2). In cohort 1, the CTLA-4 +49A/ G*A allele was found on 73% of chromosomes in patients with pSS, compared with 66% in controls (p = 0.036; odds ratio (OR) 1.41, 95% confidence interval (CI) 1.02 to 1.95). No difference in CTLA-4 CT60 allelic or genotypic distribution was observed between patients (n = 142) and controls (n = 241). In the replication cohort, the CTLA-4 +49A/G*A allele was found on 62% of chromosomes in patients with pSS, compared with 66% in controls (p = 0.30; OR 0.85, 95% CI 0.63 to 1.16). Thus, the CTLA-4 +49A/G*A allele excess among patients from cohort 1 was counterbalanced by its under-representation in cohort 2. When the results from the patients in both cohorts were pooled (n = 281), there was no difference in CTLA-4 +49A/G allelic or genotypic distribution in comparison with controls. Our results demonstrate a lack of association between CTLA-4 CT60 or +49A/G polymorphisms and pSS. Premature conclusions might have been made if a replication study had not been performed. These results illustrate the importance of case- control studies performed on a large number of patients. In fact, sampling bias may account for some contradictory results previously reported for CTLA-4 association studies in autoimmune diseases. Introduction Polymorphisms in CTLA-4, the gene encoding cytotoxic T lym- phocyte-associated antigen-4, have been widely studied in connection with genetic susceptibility to various autoimmune diseases [1], but studies have led to contradictory results in different populations. Among CTLA-4 gene polymorphisms, a G to A transition at position 49 (+49A/G) of exon 1 leads to an alanine to threo- nine amino acid substitution at codon 17 in the leader peptide (A17T), and a C to T transition at position 60 (CT60) is located within the 3'-untranslated region [2]. The G allele of +49A/G has been associated with a predisposition to many CI = confidence interval; CTLA-4 = cytotoxic T lymphocyte-associated antigen-4; OR = odds ratio; pSS = primary Sjögren syndrome; SNP = single nucleotide polymorphism. Arthritis Research & Therapy Vol 9 No 2 Gottenberg et al. Page 2 of 3 (page number not for citation purposes) autoimmune diseases (reviewed in [1]). Both polymorphisms are in linkage disequilibrium, which warrants haplotype analy- sis in studies of CTLA-4 polymorphisms. The CT60 G allele has been reported to increase susceptibility to several autoim- mune diseases [2], and a functional approach provided evi- dence for lower mRNA levels associated with the CT60 G allele [2]. Downie-Doyle and colleagues have recently reported a signif- icant association of the CTLA-4 +49A/G*A allele and of the CTLA-4 +49A/G*A allele carrier haplotypes with primary Sjö- gren syndrome (pSS), especially in patients with anti-SSA or anti-SSB antibodies, in a study including 111 Australian patients with pSS and 156 controls [3]. The aim of our study was to investigate in a large case-control study whether CTLA-4 CT60 and/or +49A/G SNPs were involved in genetic predisposition to pSS in French patients. Materials and methods Patients A first cohort of 142 unrelated patients with pSS diagnosed in accordance with the European American consensus group criteria [4] (37% without autoantibodies, 30% with anti-SSA antibodies only, and 33% with both anti-SSA and anti-SSB antibodies) and 241 healthy blood donors were genotyped for CTLA-4 CT60 and +49A/G polymorphisms. A second inde- pendent cohort of 139 patients with pSS was further geno- typed for CTLA-4 +49A/G polymorphisms in a replication study. The geographical origin and the clinico-biological char- acteristics of the patients in this second cohort were the same as those in the first. In this second cohort, 27% of patients were anti-SSA and anti-SSB negative, 35% had anti-SSA only and 38% had both anti-SSA and anti-SSB. All patients and controls were Caucasians and provided informed consent. Genotyping After the isolation of genomic DNA from peripheral blood mononuclear cells, CTLA-4 CT60 and +49A/G polymor- phisms were genotyped by restriction fragment length poly- morphism with the use of BbvI (+49A/G) and NlaIII (CT60). Statistical analysis Allelic and genotypic frequencies of CTLA-4 CT60 and +49A/ G polymorphisms were compared between patients and con- trols by using a two-sided χ 2 test. All genotyped SNPs were in Hardy–Weinberg equilibrium. CTLA-4 (+49A/G or CT60) haplotypes, constructed with the PHASE program, were also examined for association with pSS. P < 0.05 was considered significant. Results In the first cohort of patients with pSS, the A allele of the CTLA-4 +49A/G polymorphism was found on 73% of chro- mosomes in patients with pSS, in comparison with 66% in controls (p = 0.036, odds ratio (OR) 1.41, 95% confidence interval (CI) 1.02 to 1.95; Table 1). No significant difference in CTLA-4 +49A/G*A allele frequencies was observed among subgroups of patients according to their anti-SSB and/or anti- SSA status (Table 1). No difference in CTLA-4 CT60 allelic or genotypic distribution was observed between patients (n = 142) and controls (n = 241). CTLA-4 (+49A/G or CT60) hap- lotype distribution mirrored the CTLA-4 +49A/G*A allele excess among patients with pSS (A/A 48%, A/G 26%, G/G 26%, G/A 0.4%; in comparison with A/A 45%, A/G 21%, G/ G 34% among controls), leading to an excess of +49A/G*A allele carrier haplotypes among patients (p = 0.03, OR 1.41, 95% CI 1.02 to 1.95). To avoid the possibility of a false positive association of CTLA- 4 +49A/G*A with pSS as a result of the somewhat small sam- Table 1 Allellic frequencies of CTLA-4 49A/G polymorphism among patient controls CTLA-4+49A/G Allele frequencies p Odds ratio (95% CI) pSS SSA+ and SSB+ SSA+ only Ac+ Ac0 Controls (n = 241) Cohort 1 n = 142 n = 47 n = 43 n = 90 n = 52 pSS vs controls A (Thr) 208 (73) 68 (72) 61 (71) 129 (72) 79 (76) 318 (66) 0.036 1.41 (1.02–1.95) G (Ala) 76 (27) 26 (28) 25 (29) 51 (28) 25 (24) 164 (34) 0.036 0.70 (0.51–0.98) Cohort 2 n = 139 n = 52 n = 49 n = 101 n = 38 Controls (n = 241) A (Thr) 173 (62) 59 (57) 66 (67) 125 (62) 48 (63) 318 (66) NS 0.85 (0.62–1.15) G (Ala) 105 (38) 45 (43) 32 (33) 77 (38) 28 (37) 164 (34) NS 1.17 (0.86–1.60) Total n = 281 n = 99 n = 92 n = 191 n = 90 Controls (n = 241) A (Thr) 381 (68) 127 (64) 127 (69) 254 (66) 127 (71) 318 (66) NS 1.08 (0.84–1.40) G (Ala) 181 (32) 71 (36) 57 (31) 128 (34) 53 (29) 164 (34) NS 0.92 (0.71–1.19) Numbers in parentheses are percentages. pSS, primary Sjögren syndrome; Ac+, presence of anti SSB and/or anti-SSA; Ac0, absence of anti- SSA or anti-SSB antibody; CI, confidence interval; NS, not significant. Available online http://arthritis-research.com/content/9/2/R24 Page 3 of 3 (page number not for citation purposes) ple size of our first cohort, and because the CTLA-4 +49A/ G*A allele has been only marginally associated with autoim- mune diseases compared with the CTLA-4 +49A/G*G allele [1], we performed a replication study on a second independ- ent cohort of 139 patients with pSS. In this second cohort, the CTLA-4 +49A/G*A allele was found on 62% of chromosomes in patients with pSS, compared with 66% in controls (p = 0.30; OR 0.85, 95% CI 0.63 to 1.16; Table 1). Thus, the CTLA-4 +49A/G*A allele excess among patients with pSS from the first cohort was counterbalanced by its under-repre- sentation in the second cohort. When the results from the patients in both cohorts were pooled (n = 281), there was no difference in CTLA-4 +49A/G polymorphism allelic or geno- typic distribution in comparison with controls (p = 0.53, OR 1.09, 95% CI 0.84 to 1.4; Table 1). The sex ratios among patients (0.97) and controls (0.06) were different. We there- fore investigated CTLA-4 +49A/G polymorphism genotypic distribution among males and females in the control group and found that it was not statistically different (p = 0.1), thus excluding any possible gender effect. Our results therefore demonstrate a lack of association between CTLA-4 CT60 or +49A/G polymorphisms and pSS among Caucasians. Discussion The results from our first cohort were very close to those from the study of Downie-Doyle and colleagues [3], with a signifi- cant association of pSS with the +49A/G*A allele and with the +49A/G*A allele carriers haplotypes. The association observed in the first cohort, of two haplotypes bearing the same allele (CTLA-4 +49A/G*A), was actually more probably due to the statistical weight of the CTLA-4 +49A/G*A allele than to a true functional effect of two different haplotypes, bearing either CTLA-4 CT60*C or CTLA-4 CT60*T alleles, each having opposite functional effects on CTLA-4 mRNA expression [2]. In fact, our results suggest a false positive association of CTLA-4 +49A/G*A allele with pSS in the first cohort of patients. When data were pooled (cohorts 1 and 2), no signif- icant association was found with the CTLA-4 +49A/G poly- morphism in our Caucasian population of patients with pSS. This was not the consequence of different origin or different clinico-biological characteristics of the patients from the two cohorts and could only be the result of a sampling bias. Indeed, the findings observed in our first cohort of patients, as those from Downie-Doyle and colleagues [3], were unex- pected because there are only rare examples of association of the CTLA-4A/G*A allele with autoimmune diseases [5-7]. Consequently, we might have made premature conclusions if a replication study had not been performed. Conclusion Our study illustrates the necessity to include a large number of patients in genetic case-control studies. In fact, sampling bias may partly account for some contradictory results previously reported for CTLA-4 association studies in autoimmune diseases. Competing interests The authors declare that they have no competing interests. Authors' contributions JEG contributed to the study design, performed the statistical analysis and drafted the manuscript. PL and DC supervised genotyping and contributed to DNA samples collection. MA and CC performed genotyping. NC performed PHASE analy- ses. EH, XP, and JS contributed to DNA samples collection. XM and CMR supervised the study design and gave valuable advice to JEG and PL. All authors read and approved the final manuscript. Acknowledgements This study was supported by Réseau de recherche clinique INSERM. References 1. Kristiansen OP, Larsen ZM, Pociot F: CTLA-4 in autoimmune dis- eases – a general susceptibility gene to autoimmunity? Genes Immun 2000, 1:170-184. 2. Ueda H, Howson JM, Esposito L, Heward J, Snook H, Chamberlain G, Rainbow DB, Hunter KM, Smith AN, Di Genova G, et al.: Asso- ciation of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune disease. Nature 2003, 423:506-511. 3. Downie-Doyle S, Bayat N, Rischmueller M, Lester S: Influence of CTLA4 haplotypes on susceptibility and some extraglandular manifestations in primary Sjogren's syndrome. Arthritis Rheum 2006, 54:2434-2440. 4. Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, Daniels TE, Fox PC, Fox RI, Kassan SS, et al.: Classification criteria for Sjogren's syndrome: a revised ver- sion of the European criteria proposed by the American-Euro- pean Consensus Group. Ann Rheum Dis 2002, 61:554-558. 5. Djilali-Saiah I, Schmitz J, Harfouch-Hammoud E, Mougenot JF, Bach JF, Caillat-Zucman S: CTLA-4 gene polymorphism is asso- ciated with predisposition to coeliac disease. Gut 1998, 43:187-189. 6. Suppiah V, O'Doherty C, Heggarty S, Patterson CC, Rooney M, Vandenbroeck K: The CTLA4+49A/G and CT60 polymorphisms and chronic inflammatory arthropathies in Northern Ireland. Exp Mol Pathol 2006, 80:141-146. 7. Hadj Kacem H, Bellassoued M, Bougacha-Elleuch N, Abid M, Ayadi H: CTLA-4 gene polymorphisms in Tunisian patients with Graves' disease. Clin Immunol 2001, 101:361-365. . contradictory results in different populations. This case-control study sought to investigate whether CTLA-4 CT60 and/ or +49A/G polymorphisms were involved in the genetic predisposition to primary Sjögren. CTLA-4 +49A/G* A allele and of the CTLA-4 +49A/G* A allele carrier haplotypes with primary Sjö- gren syndrome (pSS), especially in patients with anti-SSA or anti-SSB antibodies, in a study including. (reviewed in [1]). Both polymorphisms are in linkage disequilibrium, which warrants haplotype analy- sis in studies of CTLA-4 polymorphisms. The CT60 G allele has been reported to increase susceptibility